CN111308100A

CN111308100A - Enzyme linked immunosorbent assay kit for detecting β -amatoxin and preparation and application thereof

Info

Publication number: CN111308100A
Application number: CN202010286487.XA
Authority: CN
Inventors: 温凯; 刘河冰; 马立才; 邢维维; 刘薇; 杨柳; 崔乃元; 丁亚芳; 聂靖东
Original assignee: Beijing Wdwk Biotechnology Co ltd
Current assignee: Beijing Wdwk Biotechnology Co ltd
Priority date: 2020-04-13
Filing date: 2020-04-13
Publication date: 2020-06-19
Anticipated expiration: 2040-04-13
Also published as: CN111308100B

Abstract

The invention discloses an enzyme linked immunosorbent assay kit for detecting β -amatoxin, and preparation and application of the kit, wherein the kit provided by the invention consists of an enzyme label plate (coated with a conjugate of a formula I β -amatoxin drug hapten and a carrier protein), an antibody working solution (containing β -amatoxin drug monoclonal antibody), an enzyme marker working solution (containing an anti- β -amatoxin drug monoclonal antibody labeled by horse radish peroxidase), a washing solution, a sample diluent, a sample extracting solution, a standard solution containing β -amatoxin with different gradient concentrations, a substrate developing solution and a stop solution.

Description

Enzyme linked immunosorbent assay kit for detecting β -amatoxin and preparation and application thereof

Technical Field

The invention belongs to the field of rapid detection of drug residues, and relates to a kit for detecting β -amatoxin, and a preparation method and application thereof.

Background

Amanitin (AMA) is a toxic cyclic peptide in the mushrooms of the Amanita genus and is a main toxin causing fatal food poisoning, β -Amanitin (β -AMA) is one of the highest-content and strongest-toxicity Amanitin, β -Amanitin causes food poisoning, is easy to misdiagnose and lacks targeted treatment, and therefore, early diagnosis is particularly important for the rescue of the Amanitin poisoning.

At present, methods for detecting amanitin toxins in biological samples such as plasma, urine and the like mainly comprise a capillary electrophoresis method, a radioimmunoassay, a high performance liquid chromatography, a mass spectrometry method and the like, wherein the liquid chromatography-tandem mass spectrometry method can realize high-flux accurate analysis of the toxins and becomes the most main method at present. The existing method has the problems of low detection flux, false positive, low sensitivity, serious matrix effect and the like, and the method with strong specificity and high sensitivity is complex to operate, expensive in instrument, not suitable for screening and detecting mass samples and cannot meet the field detection requirement.

Disclosure of Invention

The invention aims to provide an enzyme linked immunosorbent assay kit for detecting β -amatoxin, and a detection method which has high sensitivity, strong specificity and low detection cost and is suitable for screening large-batch samples.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an enzyme linked immunosorbent assay kit for detecting β -amatoxin comprises an ELISA plate, a standard substance working solution, an antibody working solution, an enzyme marker working solution, a sample diluent, a sample extracting solution, a washing solution, a substrate developing solution and a stop solution.

The ELISA plate is prepared by coating a conjugate of β -amatoxin drug hapten and carrier protein.

The β -amatoxin hapten is mL.

The β -amatoxin hapten is prepared by the following method that β -amatoxin raw material 10mg is weighed, 10mL of Dimethylformamide (DMF) is dissolved, 6.25mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 3.75mg of N-hydroxysuccinimide (NHS) are added to react for 3 hours at room temperature, 1.42mg of 6-aminocaproic acid is added to react at room temperature overnight, TLC reaction is complete, and a thin-layer preparation plate is purified to obtain the hapten (formula I).

Formula I

The carrier protein is Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), mouse serum protein (MSA), thyroxine (BCG), rabbit serum protein (RSA), hemocyanin (KLH) or Ovalbumin (OVA).

The β -amatoxin hapten and carrier protein conjugate is a product obtained by β -amatoxin hapten and carrier protein through an active ester method, and specifically comprises the following steps:

1) dissolving the β -amatoxin hapten (formula I) in DMF, adding EDC and NHS, and reacting for 2-3h at 20-25 ℃ by magnetic stirring to obtain a solution I;

wherein the ratio of the β -amatoxin hapten (formula I), the DMF, the EDC and the NHS is 2.29 mg to 0.5mL to 1 mg;

2) putting the carrier protein into 0.1M sodium bicarbonate buffer solution, stirring at 200rpm for 10min, and fully dissolving to obtain solution II; the ratio of the carrier protein to the 0.1M sodium bicarbonate buffer solution is 10 mg: 2.0 mL;

3) mixing the solution I and the solution II, specifically, dropwise adding the solution I into the solution II under the condition of 0-4 ℃ and stirring at 1000rpm, and stirring at 500rpm for 24 hours to obtain a solution III;

4) the solution III was dialyzed against PBS buffer (0.01M PBS, pH 7.2) at 4 ℃ for 3 days with stirring to obtain the β -amatoxin-coated antigen.

The β -amatoxin antibody is a specific antibody of β -amatoxin medicine.

The β -amatoxin antibody working solution is prepared by diluting a β -amatoxin monoclonal antibody by an antibody diluent by 1000 times to obtain an antibody working solution containing a β -amatoxin drug monoclonal antibody.

The antibody diluent is a PBS buffer solution containing Proclin300 and Triton X-100; the solvent of the antibody diluent is deionized water, the solutes are anhydrous disodium hydrogen phosphate, dihydrate sodium dihydrogen phosphate, sodium chloride, potassium chloride, Proclin300 and Triton X-100, and the concentrations of the solutes in the PBS buffer are 1.072 g/L, 0.59g/L, 8.5g/L, 0.4g/L, 200 muL/L and 500 muL/L respectively.

The enzyme marker working solution is obtained by diluting an anti-antibody of an anti- β -amatoxin drug monoclonal antibody marked by horseradish peroxidase by 500 times with an anti-antibody diluent.

The kit comprises an anti-antibody diluent, a reagent kit and an enzyme label plate, wherein the anti-antibody diluent is a PBS buffer solution containing calf serum and Proclin300, the solvent of the antibody diluent is deionized water, the solutes are anhydrous disodium hydrogen phosphate, dihydrate sodium dihydrogen phosphate, sodium chloride, potassium chloride, calf serum and Proclin300, the concentrations of the solutes in the PBS buffer solution are respectively 1.072 g/L, 0.6 g/L, 16 g/L, 0.4g/L, 50 mL/L and 200 muL/L, and in the kit, the conjugate of the β -amatoxin drug hapten and the carrier protein is coated on the enzyme label plate.

In the kit, the specific antibody of the β -amatoxin drug is the β -amatoxin monoclonal antibody.

In the kit, the β -amatoxin monoclonal antibody consists of a heavy chain and a light chain, wherein the amino acid sequence of the variable region of the heavy chain can be shown as the sequence 1 in a sequence table, and the amino acid sequence of the variable region of the light chain can be shown as the sequence 2 in the sequence table.

In the kit, the solvents of the 6 standard substance working solutions are PBS buffer solutions containing light stabilizers and bovine serum albumin, the solutes are β -amatoxin, the concentrations of the solutes in the 6 standard substance working solutions are respectively 0 mug/L, 1 mug/L, 3 mug/L, 9 mug/L, 27 mug/L and 81 mug/L, the solvents of the PBS buffer solutions are deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizers and bovine serum albumin, the concentrations of the solutes in the PBS buffer solutions are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L and 0.1 g/L, and the pH value is 7.2.

In the kit, the sample diluent is a PBS buffer solution containing a light stabilizer, bovine serum albumin and a surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L, 0.1 g/L and 0.1 g/L, and the pH value is 7.2.

In the kit, the sample extracting solution is PBS buffer solution containing a light stabilizer, bovine serum albumin and a surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L, 0.1 g/L and 0.2g/L, and the pH value is 7.2.

In the kit, the washing solution is PBS buffer solution containing Tween 20 and Proclin 300; the solvent of the washing solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentrations of the solutes in the PBS buffer solution are 23.2 g/L, 2.0 g/L, 64 g/L, 0.036 g/L, 20 mL/L and 300 muL/L respectively.

In the kit, the substrate color development liquid is a mixed aqueous solution of 1.0 g/L carbamide peroxide, 5.0 g/L sodium acetate, 0.5 g/L light stabilizer, 2.5 mL/L phosphoric acid and 5.0 g/L tetramethyl benzidine, and the solvent is deionized water.

In the kit, the stop solution is 0.05 mol/L sulfuric acid aqueous solution.

Another object of the present invention is to provide a method for detecting β -amatoxin in a sample, comprising the steps of:

1) pretreating a sample to obtain a solution of the sample;

2) detecting with any one of the above-mentioned kits;

3) and analyzing the detection result.

In the method, the detection by using the kit comprises the following steps of adding a standard working solution or a solution of the sample into an enzyme label plate coated with a conjugate of the β -amatoxin drug hapten and a carrier protein, then adding a specific antibody solution containing β -amatoxin drug, washing and patting dry after incubation, adding the enzyme-labeled anti-antibody, developing by using a substrate, stopping, and measuring the light absorption value by using an enzyme label apparatus.

In the above method, the method for pretreating the sample specifically comprises the following steps:

accurately weighing 1+ -0.01 g (or mL) of sample, adding 5mL of sample extract, whirling at high speed for 1min at room temperature (25 + -2 deg.C), and centrifuging at 4000 g for 5min to obtain sample solution. And adding 200 mu L of sample solution into 200 mu L of sample diluent, whirling at a high speed for 1min, and taking 50 mu L of supernatant to analyze.

The detection principle of the kit is that an indirect competitive ELISA method capable of detecting β -amatoxin is adopted, an ELISA plate is coated by a conjugate of β -amatoxin drug hapten and carrier protein, β -amatoxin drug monoclonal antibody is used as a first antibody, an anti-antibody of β -amatoxin drug monoclonal antibody marked with Horse Radish Peroxidase (HRP) is used as a second antibody, substrate liquid is added for color development reaction, and the concentration of 0.05 mol/L H mol is used as the second antibody₂SO₄The reaction was stopped and the absorbance at 450 nm was measured to detect β-amatoxin.

The analysis process of the detection result provided by the invention comprises the following steps:

the absorbance average (B) of the standard working solution of each concentration obtained was divided by the absorbance value (B0) of the first standard solution (0 standard) and multiplied by 100%, i.e., the percent absorbance value. The calculation formula is as follows: percent absorbance value (%) - (B/B0). times.100%

And (3) taking the half-logarithmic value of the concentration (mu g/L) of the β -amatoxin standard working solution as an X axis and the percent absorbance value as a Y axis, drawing a standard curve graph, calculating the percent absorbance value of the sample solution by the same method, and reading the β -amatoxin content in the sample from the standard curve corresponding to the concentration of each sample.

The analysis of the detection result in the invention can also adopt a regression equation method to calculate the concentration of the sample solution.

The analysis of the detection result can also utilize computer professional software, the method is more convenient for the rapid analysis of a large number of samples, and the whole detection process can be completed within 1.5 h in a short time.

Experiments prove that the kit can detect β -amatoxin in serum and urine, the detection limit of β -amatoxin in serum and urine is 10 mug/kg, the inter-batch variation coefficient is less than 10%, the intra-batch variation coefficient is less than 15%, and the stability is good.

The β -amatoxin detection kit provided by the invention can be used for detecting β -amatoxin in serum and urine, has the advantages of simple and rapid operation, high sensitivity, strong specificity and strong accuracy, and has great value for rapid detection of food poisoning.

Drawings

FIG. 1 β -Amanit standard graph.

FIG. 2 is a comparison of the results of the detection by the present kit with LC-MS/MS.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of enzyme-linked immunosorbent assay kit for detecting β -amatoxin

The kit comprises an enzyme label plate (coating β -a conjugate of an amatoxin drug and a carrier protein), an antibody working solution (containing β -an amatoxin drug monoclonal antibody), an enzyme marker working solution (containing an anti- β -amatoxin drug monoclonal antibody marked by horseradish peroxidase), a washing solution, a sample diluent, a sample extracting solution, a working solution containing different gradient concentrations of β -amatoxin standard substance, a substrate developing solution and a stop solution.

The preparation method comprises the following steps:

firstly, preparation of enzyme label plate

1.β preparation of amatoxin-coated antigen

(1) β preparation of amatoxin hapten

Weighing β -amatoxin raw material 10mg, dissolving 10ml DMF, stirring, adding 6.25mg EDC,3.75mg NHS, reacting for 3h at room temperature, adding 1.42mg 6-aminocaproic acid, reacting overnight at room temperature, TLC reacting completely, purifying with thin layer preparation plate to obtain hapten (formula I):

formula I

(2) β preparation of amatoxin-coated antigen

Dissolving 2.29 mg of β -amatoxin hapten with 0.5mL of DMF, stirring at 200rpm for 10min, adding 31.0mg of EDC and 1.0mg of NHS to dissolve, stirring at room temperature (500 rpm) to activate for 2-3h to obtain solution I, weighing OVA10 mg to dissolve in 3.5 mL0.1M sodium bicarbonate solution, stirring at 200rpm for 10min to fully dissolve, cooling in an ice bath at 0-4 ℃, stirring at 1000rpm, dropwise adding (1 mL/min) the solution I reaction solution, stirring at 500rpm for 24h to react, filling the reaction product into a distilled water washing dialysis bag (10 cm), washing with 1L of 0.01M PBS (1 x, pH 7.2) at 4 ℃, stirring (100 rpm) to dialyze for 3d, changing the solution every day (once in the morning, in the evening), changing the solution for 9 times in total, and centrifuging the dialysis product at rpm for 6min to obtain β -amatoxin coating.

2. Preparation of ELISA plates

Diluting the obtained β -amatoxin coated antigen to 10.0 mu g/mL by using a coating buffer solution, adding 100 mu L of the coated antigen into each hole, incubating for 2 h at 37 ℃, pouring out the coating solution, washing for 2 times by using a washing solution diluted by 20 times, each time for 30 s, patting dry, then adding 150 mu L of a sealing solution into each hole, incubating for 1 h at 37 ℃, pouring out the liquid in the holes, drying to obtain the elisa plate coated with the coated antigen (conjugate of β -amatoxin drug hapten and carrier protein), and preserving in a vacuum sealing manner by using an aluminum film.

Coating buffer solution: 0.03 mol/L sodium carbonate buffer solution with the pH value of 9.6;

sealing liquid: contains 50 g/L sucrose, 2.5 g/L casein, 0.5% calf serum, and 3 ‰ sodium azide in 0.2 mol/LpH7.7 phosphate buffer solution.

Preparation of antibody working solution

1.β -preparation of amatoxin monoclonal antibody

(1) β preparation of amatoxin immunogen

Dissolving 1.54mg β -amatoxin hapten with 0.5ml DMF, stirring at 200rpm for 10min, adding 1mg EDC, dissolving, adding NHS 1mg, stirring at room temperature (500 rpm) for 2-3h, obtaining solution I, weighing BSA 10mg, dissolving in 2ml 0.1M sodium bicarbonate solution, stirring at 200rpm for 10min, fully dissolving, cooling in ice bath at 0-4 ℃, stirring at 1000rpm, adding dropwise (1 ml/min) solution I reaction solution, stirring at 500rpm for reaction for 24h, filling the reaction product into a distilled water washing dialysis bag (10 cm), 1L0.01M PBS (1 x, pH 7.2), stirring at 4 ℃ (100 rpm) for dialysis for 3d, changing the solution every day (once in the morning, at night), changing the solution for 9 times, centrifuging at 5000rpm for 6min, 1.5 ml/tube, numbering the antigen, storing at-20 ℃ for later use, and obtaining the amatoxin immunogen of β -amatoxin.

(2) Animal immunization

Dissolving the prepared β -amatoxin immunogen by normal saline according to 100 mu g/mouse, uniformly mixing the immunogen and Freund's complete adjuvant in equal volume, injecting and immunizing Balb/c female mice with 6-8 weeks old by subcutaneous injection on the neck and back, uniformly mixing the immunogen and Freund's incomplete adjuvant in equal volume on 7 th, 14 th and 28 th days after primary immunization, performing additional immunization once respectively, and performing additional immunization once by using 100 mu g/mouse of immune complex 3 days before fusion without adding Freund's adjuvant.

(3) Cell fusion and cloning

Mixing splenocytes of immunized mice with myeloma cells of mice (SP 2/0) in logarithmic growth phase, slowly adding preheated fusion agent (PEG 4000) within 45 s for fusion, suspending with HAT medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate at 37 deg.C and 5% CO₂Culturing in an incubator, half-changing the culture medium with HT after 5 days, and completely changing the culture medium after 9 days.

After cell fusion, when the cell grows to 1/4 of culture hole area, screening hybridoma cell by step screening method, adopting indirect ELISA method to coat enzyme label plate with coating antigen (the optimum coating concentration and positive serum dilution are titrated by square matrix method in advance), adding culture supernatant of tested hole, incubating, washing, adding goat anti-mouse IgG-HRP and IgM-HRP, OPD to make color reaction, screening the screened positive hole by indirect competition ELISA method, mixing cell supernatant with β -amatoxin of 100 microgram/mL, water bathing at 37 deg.C for 30 min, adding into coated enzyme label plate, using PBS to replace β -amatoxin as control, the rest steps are the same as above, if OD after β -amatoxin blocking is OD 25-amatoxin blocking₄₅₀And (3) judging the wells to be positive when the nm value is reduced to below 50% of the control wells, and subcloning the wells which are positive after 2-3 detections by using a limiting dilution method immediately.

(4) Preparation and purification of monoclonal antibodies

Carrying out expanded culture on the hybridoma after 2-3 times of subcloning and strain establishment, collecting supernatant, measuring titer by using indirect ELISA, and freezing and storing; injecting 0.5 mL/mouse of liquid paraffin into the abdominal cavity of a Balb/c mouse aged 8-10 weeks, injecting 1-2 × 10 hybridoma cells into the abdominal cavity after 7-10 days⁵Ascites was extracted 7 to 10 days later. Collecting cell supernatant or ascites, and measuring titer by indirect ELISA (P/N for measuring titer)>2.1 on cellsThe maximum dilution times of the clear fluid or the ascites fluid) shows that the titer of the cell supernatant is 1: 10000, the titer of the ascites fluid is 1: 60000, and then the cell supernatant is purified by an octanoic acid-saturated ammonium sulfate method, and the supernatant is taken to obtain the purified monoclonal antibody of the β -amatoxin drug.

The chessboard method is used for measuring the potency of the monoclonal antibody, and the result shows that the potency of the β -amatoxin drug monoclonal antibody is 1: 200000, and the half inhibition amount (IC)₅₀) It was 1.7. mu.g/L.

Through detection, the amino acid sequence of the variable region of the heavy chain of the β -amatoxin monoclonal antibody is shown as the sequence 1 in the sequence table, and the amino acid sequence of the variable region of the light chain of the β -amatoxin monoclonal antibody is shown as the sequence 2 in the sequence table.

2. Preparation of antibody working solution

Diluting the obtained β -amatoxin drug monoclonal antibody 1000 times with antibody diluent to obtain β -amatoxin drug monoclonal antibody working solution.

Thirdly, preparation of enzyme marker working solution

1. Preparation of anti-antibodies

The obtained β -amatoxin drug monoclonal antibody is used as immunogen, and goat is used as immune animal, so as to obtain the goat anti-mouse anti-antibody of the β -amatoxin drug monoclonal antibody.

2. Preparation of horse radish peroxidase-labeled anti-antibody

Coupling the anti-antibody of the monoclonal antibody of the β -amatoxin drug obtained in the step 1 with horseradish peroxidase (HRP) by a modified sodium periodate method, and the following steps:

dissolving 8 mg of horseradish peroxidase in 2mL of distilled waterPerforming the following steps; adding 100 mmol/L NaIO prepared now₄Stirring and reacting the solution for 20 min at room temperature, wherein the solution is 0.4 mL; dialyzing with 1 mmol/L acetate buffer solution at 4 deg.C overnight; removing excess NaIO₄Simultaneously reducing the self-coupled enzyme, adding 40 μ L PBS buffer (pH 8.6, 0.5 mol/L) and 2.0 mL PBS buffer (pH 8.6, 5 mol/L) containing 16 mg monoclonal antibody against β -amatoxin drug, stirring at room temperature for 4 hr, adding 0.1 mL 1 mol/L NaBH prepared₄Reacting the aqueous solution at 4 ℃ for 4 hours, purifying and storing.

3. Preparation of enzyme-labeled working solution

And (3) diluting the antibody of the monoclonal antibody of the β -amanitin drug which is marked by the horseradish peroxidase by 500 times by using an anti-antibody diluent to obtain an enzyme marker working solution of the anti-antibody of the monoclonal antibody of the β -amanitin drug which is marked by the horseradish peroxidase.

The anti-antibody diluent is a PBS (phosphate buffer solution) containing calf serum and Proclin 300; the solvent of the antibody diluent is deionized water, the solutes are anhydrous disodium hydrogen phosphate, dihydrate sodium dihydrogen phosphate, sodium chloride, potassium chloride, calf serum and Proclin300, and the concentrations of the solutes in the PBS buffer are 1.072 g/L, 0.6 g/L, 16 g/L, 0.4g/L, 50 mL/L and 200 muL/L respectively.

Fourth, preparation of standard substance working solution

The kit also comprises 6 standard substance working solutions, wherein solvents of the 6 standard substance working solutions are PBS buffer solutions containing light stabilizers and bovine serum albumin, solutes are β -amatoxin, the concentrations of the solutes in the 6 standard substance working solutions are respectively 0 mug/L, 1 mug/L, 3 mug/L, 9 mug/L, 27 mug/L and 81 mug/L, the solvents of the PBS buffer solutions are deionized water, disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizers and bovine serum albumin, the concentrations of the solutes in the PBS buffer solutions are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L and 0.1 g/L, and the pH value is 7.2.

Preparation of five, other reagents

The kit may further contain a sample diluent and/or a sample extract and/or a washing solution and/or a substrate developing solution and/or a stop solution.

The sample diluent is a PBS buffer solution containing a light stabilizer, bovine serum albumin and a surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L, 0.1 g/L and 0.1 g/L, and the pH value is 7.2.

The sample extracting solution is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L, 0.1 g/L and 0.2g/L, and the pH value is 7.2.

The washing solution is PBS buffer solution containing Tween 20 and Proclin 300; the solvent of the washing solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentrations of the solutes in the PBS buffer solution are 23.2 g/L, 2.0 g/L, 64 g/L, 0.036 g/L, 20 mL/L and 300 muL/L respectively.

The substrate color developing solution is a mixed aqueous solution of 1.0 g/L carbamide peroxide, 5.0 g/L sodium acetate, 0.5 g/L light stabilizer, 2.5 mL/L phosphoric acid and 5.0 g/L tetramethyl benzidine, and the solvent is deionized water.

The stop solution is 0.05 mol/L sulfuric acid aqueous solution.

Example 2 and example 1 methods of Using the kits

First, pretreatment of sample

1. Pretreatment of serum and urine samples

Second, detection Using the kit of example 1

1. Inserting an enzyme label plate into an enzyme label plate frame, recording the positions of each standard product and each sample, making 3 samples in parallel, sealing unused enzyme label plates by using self-sealing bags, and immediately storing in an environment at 2-8 ℃;

2. respectively adding 50 mu L of each standard substance working solution or sample solution into the corresponding standard substance or sample hole;

3. adding 50 mu L of antibody working solution into each plate hole;

4. covering a cover plate membrane, slightly oscillating the ELISA plate for 10 s, fully and uniformly mixing, and reacting for 30 min at room temperature (25 +/-2 ℃) in a dark place;

5. uncovering the plate film, pouring out liquid in the plate holes, adding 260 mu L of washing working solution (the washing solution is diluted by 20 times by deionized water) into each hole, and fully washing for 3-4 times, wherein each time of soaking is 15-30 s; (ii) a

6. Pouring out liquid in the plate hole, pouring the enzyme label plate on absorbent paper, and patting dry;

7. adding 100 mu L of enzyme marker working solution into each plate hole; covering a cover plate film, slightly oscillating the ELISA plate for 10 s, fully and uniformly mixing, and reacting for 30 min at room temperature (25 +/-2 ℃) in a dark place;

8. repeating the step 5-6;

9. immediately adding 100 μ L of substrate color developing solution A, B mixed solution (substrate color developing solution A and substrate color developing solution B are mixed according to volume ratio of 1: 1) into each well, covering with a cover plate membrane, and reacting for 15 min in dark place;

10. uncovering the microplate membrane, adding 50 mu L of stop solution into each plate hole, slightly oscillating the ELISA plate for 10 s, and fully and uniformly mixing;

11. and reading the absorbance values of the ELISA plate at the dual wavelengths of 405 nm and 630 nm by using an ELISA reader within 5min after termination.

Thirdly, analyzing the detection result

1. Calculating percent absorbance value

The average absorbance value of each standard (or sample to be measured) is divided by the absorbance value of zero standard (standard with concentration of 0 mug/L) and multiplied by 100 percent, so as to obtain the percentage of absorbance corresponding to each standard (or sample to be measured), namely the percent absorbance value.

Percent absorbance = B/B₀×100%

Wherein: b-mean absorbance value of standard (or sample); b is₀Average absorbance values of standards at a concentration of 0. mu.g/L.

2. Making a standard curve

The percent absorbance values of the standards were plotted as ordinate and the β -amatoxin concentration (μ g/L) in the working solution of each standard as abscissa to plot a standard curve, and origin8.0 (OriginLab corp., Northampton, MA, USA) was used for non-linear fitting analysis to form a four-parameter fitting curve:

y=(A-D)/[1+(x/C)B]+D

wherein y is the percentage of absorbance; x is the concentration of the substance to be detected; a, B, C and D are the four parameters of the standard curve.

Through test data, the standard curve equation of β -amatoxin is that Y = -0.003+ (2.309+0.003)/(1+ (x/5.505) ^1.03), and the linear correlation R²Is 0.999.

The standard graph is shown in fig. 2.

3. Calculating the β -amatoxin content in the sample

Substituting the percent absorbance value of the sample to be detected into the standard curve to obtain the corresponding residual concentration of the sample to be detected, and multiplying the residual concentration by the dilution times of the corresponding sample to obtain the actual content of β -amatoxin in the original sample to be detected.

Example 3 and example 1 detection of specificity, detection Limit, accuracy, precision of the kits

Firstly, specific test of the kit:

β -amatoxin ELISA kit specificity through the corresponding substances were determined by cross reaction test.

Respectively diluting β -amatoxin and its analogs (β -amatoxin and phallotoxin) in series, and respectively performingExample 2 the procedure was carried out to replace "β -amatoxin standard working solution" with β -amatoxin and its analogue serial dilutions, to prepare standard curves, and to find out respective 50% Inhibitory Concentrations (IC) on the curves₅₀) The specific method comprises obtaining β -amatoxin concentration (μ g/L) with ordinate value equal to 50%, i.e. IC₅₀The cross-reactivity of the kit to β -amatoxin and each of the commonly used muricides was calculated using the following formula:

cross-reactivity (%). times (β -amatoxin concentration causing 50% inhibition/β -amatoxin analogue concentration causing 50% inhibition) × 100%.

The results are shown in Table 1.

TABLE 1 specificity of the kit

Name (R)	Cross reaction Rate (%)
		β Amanit venom peptide	100.0
α Amanit venom peptide	<1%
		Coprinus toxicide	<1%

Experiments show that the kit has better specificity to β -amatoxin, namely the kit can detect β -amatoxin.

Second, detection limit determination of the kit

A blank sample of serum or urine (negative in LC-MS/MS) was sampled and tested according to the method of example 2, the values were determined from the standard curve, the average value was calculated, and the lowest limit of detection (LOD) was calculated by adding 3 times the standard deviation. The results are shown in Table 2.

TABLE 2 blank sample determination results (μ g/L)

The result shows that in order to prevent the occurrence of false positive, the detection limit of the kit on β -amatoxin in serum and urine can be determined to be 10 mug/L.

Thirdly, testing the accuracy and precision of the kit

Taking serum and urine blank samples (LC-MS/MS detection negative) to be respectively pretreated according to the method in the first step of the embodiment 2, and then adding β -amatoxin standard substance to the required concentration of 10 mu g/kg and 20 mu g/kg to obtain a detection sample solution.

The detection is carried out by using 3 kits of different batches, each experiment is repeated for 5 times, and the coefficient of variation is calculated respectively. The results are shown in Table 3, respectively.

The method for calculating the intra-batch variation coefficient comprises the following steps: intra-batch coefficient of variation = coefficient of variation for each parallel sample in the same assay.

The method for calculating the inter-batch variation coefficient comprises the following steps: the inter-batch coefficient of variation = coefficient of variation of measurement results of the same sample in different batches, and the average value thereof is taken.

TABLE 3 accuracy and precision

The result shows that the recovery rate of each addition concentration of all samples is between 80 and 120 percent. The variation coefficient in batch of each addition concentration is lower than 10%, and the variation coefficient between batches is lower than 15%.

Fourthly, the detection result of the kit is compared with the detection result of LC-MS/MS

The method of example 2 is used to detect serum and urine samples, and the detection results of LC-MS/MS are respectively used for confirmation and comparison.

The concentration of β -amatoxin measured by the kit is taken as an X axis, the concentration of β -amatoxin measured by LC-MS/MS is taken as a Y axis, a scatter diagram is drawn, the results of the two methods are subjected to linear analysis, the result is shown in figure 2, the regression equation is Y =1.003X-0.022, and the method established by the invention has good consistency with the LC-MS/MS detection result.

Fifth, shelf life test of kit

The storage condition of the kit in the embodiment 1 is 2-8 ℃, the maximum absorbance (zero standard), the 50% inhibition concentration and the β -amatoxin addition actual measurement value of the kit are all within the normal range after 12 months of measurement, the kit is placed for 8 days at 37 ℃ to carry out an accelerated aging test considering that abnormal storage conditions occur in the transportation and use processes, and the result shows that all indexes of the first step to the fourth step of the kit completely meet the requirements, the kit is placed for 8 days at-20 ℃ considering that the freezing condition of the kit occurs, and all indexes of the first step to the fourth step completely meet the requirements.

Sequence listing

<110> Beijing Weideweikang Biotech Ltd

<120> enzyme linked immunosorbent assay kit for detecting β -amatoxin and preparation and application thereof

<141>2020-04-13

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>118

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Gln Leu Val Glu Ser Gly Pro Gly Val Leu Val Ala Pro Ser Gln Ser

1 5 10 15

Leu Ser Thr Ile Cys Thr Val Asp Ser Leu Gly Ser Phe Thr Tyr Gly

20 25 30

Val Asn Trp Val Arg Gln Pro Pro Gly Gly Ser Thr Asp Lys Glu Trp

35 40 45

Leu Gly Trp Gly Leu Ile Trp Gly Gly Glu Tyr Asn Ala Leu Lys Lys

50 55 60

Arg Ile Ser Arg Asp Leu Ser Ser Lys Asn Ser Asp Ser Gln Val Phe

65 70 75 80

Met Asn Asp Asp Ser Gln Leu Thr Thr Ala Arg Tyr Tyr Gly Ile Cys

85 90 95

Ala Arg Asp Lys Tyr Trp Asn Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210>2

<211>113

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

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Asp Ile Val Gln Ser Pro Asp Ile Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ser Met Cys Arg Gln Ser Leu Thr Val Asn Gly His

20 25 30

Ser Asn Tyr Leu His Ser Ser Trp Tyr Leu Gln Gln Pro Lys Gly Ser

35 40 45

Pro Lys Leu Ile His Lys Val Ser Leu Asn Arg Phe Val Ser Pro Asp

50 55 60

Ser Gly Gly Ser Arg Leu Phe Glu Gly Ser Thr Asp Gly Phe Thr Leu

65 70 75 80

Ser Arg Val Glu Ala Asp Leu Glu Gly Val Tyr Phe Ser Thr Cys Ser

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Gln His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile

100 105 110

Lys

Claims

1. An enzyme linked immunosorbent assay kit for detecting β -amatoxin comprises a β -amatoxin detection ELISA plate, β -amatoxin series standard substance working solution, β -amatoxin antibody working solution, enzyme marker working solution, sample diluent, sample extracting solution, washing solution, substrate developing solution and stop solution, and is characterized in that the β -amatoxin ELISA plate is represented by formula I:

formula I

β -amatoxin drug hapten and carrier protein conjugate coating, the β -amatoxin antibody is β -amatoxin drug specific antibody.

2. The enzyme-linked immunoassay kit for detecting β -amatoxin according to claim 1, wherein the preparation method of β -amatoxin hapten comprises the steps of weighing 10mg of raw material, dissolving 10mg of LDMF, adding 6.25mg of EDC, reacting 3.75mg of NHS at room temperature for 3h, adding 1.42mg of 6-aminocaproic acid, reacting overnight at room temperature, completing TLC reaction, and obtaining β -amatoxin hapten.

3. The enzyme-linked immunoassay kit for detecting β -amatoxin according to claim 1, characterized in that the specific antibody of β -amatoxin drug is the β -amatoxin monoclonal antibody, the β -amatoxin monoclonal antibody consists of a heavy chain and a light chain, the amino acid sequence of the variable region of the heavy chain can be shown as sequence 1 in the sequence table, and the amino acid sequence of the variable region of the light chain can be shown as sequence 2 in the sequence table.

4. The ELISA kit for detecting β -amatoxin according to claim 1 or 3, wherein the β -amatoxin antibody working solution is prepared by diluting β -amatoxin monoclonal antibody with antibody diluent 1000 times to obtain β -amatoxin drug monoclonal antibody working solution;

5. The ELISA kit for detecting β -amanitin according to claim 1, wherein the enzyme label working solution is obtained by diluting 500 times an anti-antibody of an anti- β -amanitin drug monoclonal antibody labeled by horseradish peroxidase with an anti-antibody diluent;

6. The enzyme-linked immunoassay kit for detecting β -amatoxin according to claim 1, wherein the β -amatoxin series standard working solution is 6 β -amatoxin standard working solutions, the solvent of the solution is PBS buffer solution containing light stabilizer and bovine serum albumin, the solute of the solution is β -amatoxin, the concentrations of the solutes in the 6 standard working solutions are 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L and 81 μ g/L respectively, the solvent of the PBS buffer solution is deionized water, the solutes are sodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer and bovine serum albumin, the concentrations of the solutes in the PBS buffer solution are 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L and 0.1 g/L respectively, and the pH value is 7.2.

7. The enzyme-linked immunoassay kit for detecting β -amatoxin according to claim 1, wherein the kit further comprises a sample diluent, a sample extract, a washing solution, a substrate developing solution and a stop solution;

the sample diluent is a PBS buffer solution containing a light stabilizer, bovine serum albumin and a surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L, 0.1 g/L and 0.1 g/L, and the pH value is 7.2;

the sample extracting solution is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L, 0.1 g/L and 0.2g/L, and the pH value is 7.2;

the washing solution is PBS buffer solution containing Tween 20 and Proclin 300; the solvent of the washing solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentrations of the solutes in the PBS buffer solution are 23.2 g/L, 2.0 g/L, 64 g/L, 0.036 g/L, 20 mL/L and 300 muL/L respectively;

the substrate color developing solution is a mixed aqueous solution of 1.0 g/L carbamide peroxide, 5.0 g/L sodium acetate, 0.5 g/L light stabilizer, 2.5 mL/L phosphoric acid and 5.0 g/L tetramethyl benzidine, and the solvent is deionized water;

the stop solution is 0.05 mol/L sulfuric acid aqueous solution.

8. A method for detecting β -amatoxin residue in a sample, comprising the steps of:

1) pretreating a sample;

2) detecting with the kit of claim 1;

3) and analyzing the detection result.

9. The method for detecting β -amatoxin residue in a sample according to claim 8, wherein the detection with the kit comprises the steps of adding a standard working solution or a solution of the sample to an ELISA plate coated with the conjugate of β -amatoxin drug hapten and carrier protein, adding a specific antibody of β -amatoxin drug, washing and drying after incubation, adding the enzyme-labeled anti-antibody, developing with an alkaline phosphatase substrate, terminating, measuring the absorbance with an enzyme reader, developing with a horseradish peroxidase substrate, terminating, and measuring the absorbance with an enzyme reader.

10. The method for detecting β -amatoxin residue in a sample according to claim 8, wherein β -amatoxin in serum and urine can be detected by using the kit, the detection limit of β -amatoxin in serum and urine is 10 μ g/kg, the inter-batch variation coefficient is less than 10%, and the intra-batch variation coefficient is less than 15%.